Pneumonic plague results from the inhalation of the bacterium Yersinia pestis. Due to its lethality, rapid disease progression, and ability to be disseminated via aerosolization, Y. pestis is categorized by the U.S. Centers for Disease Control as one of the six Category A agents most likely to be used during a biological terrorist attack. Despite its lethality and history as a human pathogen, very little is known regarding pneumonic plague pathogenesis, as a majority of work has focused on bubonic infection and with less virulent or avirulent strains. In a mouse infection model pneumonic plague was shown to progress in two distinct phases. During the first 36 hours of disease bacteria replicate to large burdens in the lung in the absence of host inflammation or disease pathology. After 36-48 hours, an intense inflammatory phase is initiated, resulting in severe bronchopneumonia. In order to survive, Y. pestis must adapt to these two very different environments in the mammalian lung, initially evading/suppressing immunity and subsequently withstanding the host inflammatory onslaught. The ultimate goal of this application is to obtain a more complete understanding of the progression of pneumonic plague by identifying bacterial virulence factors and target host cells. Y. pestis expressing green fluorescent protein (GFP) will be used to infect mice intranasally, and at different times throughout infection lungs will be harvested and analyzed by flow cytometry to identify cell populations targeted by Y. pestis, and how those populations change over time. To identify bacterial factors involved in disease progression, quantitative real-time PCR will be used to evaluate the transcription of 288 Y. pestis genes previously shown to be regulated at 48 hours post-infection. The transcription of genes of interest will be evaluated at multiple time points during infection. Genes that are highly regulated or demonstrate differential regulation between phases of infection will be deleted, and mutant strains evaluated in a mouse model of infection to determine their role in pneumonic plague. PUBLIC HEALTH RELEVANCE: Inhalation of the bacterium Yersinia pestis results in pneumonic plague, a rapidly progressing disease that is 100 percent fatal if antibiotics are not delivered in a timely manner. Its lethality and ability to be disseminated as infectious aerosol droplets drive fears that Y. pestis may be used as a biological weapon. A lack of understanding of disease progression has been a significant barrier to the development of improved treatment options. The work proposed here seeks to identify Y. pestis factors important for disease as well as target cells in the lung for the identification of novel drug and vaccine targets.